Success with Direct Sequening of Bisulfite Pcr Products - (Mar/24/2006 )
HI, I just started using the Epitect Kit from Qiagen, however I was only able to recover 5% of my initial 1ug gDNA. I sheared my gDNA between 200 to 700bp and used 1ug of DNA for conversion without addition of carrier RNA. Anyway to increase the yield?
I would suggest to not shear the DNA. I begin with 2ug DNA (phenol-chloroform cleaned though may not be 100% essential) and elute in 30ul, which I then dilute 1:3, so usually have enough for a little less than 45 PCR's. This is roughly equal to 45ng of pre-converted dsDNA per PCR. If you used 1ug instead of 2 you should still get a reasonable amount of DNA if you diluted 1:2 even.
What method of quantitation do you recommend for the converted DNA? Is Nanodrop ssDNA function accurate? What is a good way to view the converted DNA for checks on any DNA degradation?
I don't quantify the DNA after conversion. I'm not sure you can do an OD to get an estimate of the degradation. Perhaps an agarose gel may be more useful. Run some unconverted DNA alongside it to see any difference. If you are only going to perform a PCR for sequencing or MSP, don't bother quantitating it.
Thanks Davo for your explanation, but I was not wrong, I understood well that on the reverse sequence, I have to consider the G/A peaks which corresponds to the C/T peak on the forward strand. However, I still do not have the same methylation status, I observe methylation when sequencing with the forward primer and no methylation with the reverse primer, from the same PCR fragment.
And I have now another problem : I started to clone my PCR fragments and sequence several clones. But again, I almost have no methylation (less than 10 %) on the clones for sample where I observed about 50% methylation by direct sequencing. I did so far just 2 samples but it is quite scary to see that. Anyone knows why ???? I cloned my PCR products in pGEMT easy vector (Promega).
I'm starting out on MSP but is unsure of the primer design. Using a hypermethylated gene I design the PCR primers at the promoter region that contain CpG islands. For the met primers I convert the original DNA sequence to a fully methylated sequence before designing the primers. But I don't see any bands, wonder if my PCR design is wrong or the DNA is not fully converted.
Your primers for the methylated reaction should be designed around all CpG C's as C's, and all non CpG C's as T's. If you have that right then you may need to optimise your PCR reaction. Have you tried different amount of template and magnesium? Lower annealing temps in the PCR cycles may also help. And yes this will depend on your template being converted. How are you doing the bisulfite conversion? Most of the commercially available kits do a good job at the conversion.
I'm using the Epitech kit from Qiagen, I did 2 rounds of consecutive conversion and got at least 50% recovery. As for the primers I used Methylprimer to design my primers. I'm wondering if you could send me your MSP PCR condition and cycling program for my reference? I'm using the pfuTurbo Cx Hotstart polymerase for PCR.